Autotransporter (AT) proteins are a large and highly diverse family of secreted and outer membrane proteins, that are taxonomically widespread amongst Gram-negative bacteria. These proteins have a conserved set of architectural domains that consists of an N-terminal signal sequence which directs protein secretion across the inner membrane, a translocation (β) domain which is embedded into the outer membrane, and a functional (α) domain which is either presented on the cell surface or released into the external milleu. The AIDA-I-type adhesins make up one of the largest group of AT proteins, and play a key role in bacterial pathogenesis, by promoting virulent phenotypes such as adhesion, cell aggregation and biofilm formation. This work focuses on characterising Antigen 43 (Ag43) from two pathogenic E. coli strains: uropathogenic E. coli (UPEC), main cause of urinary tract infections, and enterohemorrhagic E. coli (EHEC), causative agent of severe foodborne diseases. The study is targeted towards structurally and functionally characterising the different Ag43 homologues and investigating the mechanisms involved in AT processing and self-recognition, which promote different virulence functions. The crystal structures of the α-domain of both UPEC Ag43 and EHEC Ag43 have revealed self-association mechanisms that differ from that of the well-characterised Ag43a. By investigating the structure-function relationship of the α-domain of Ag43 homologues, we aim to determine avenues to inhibit AT mediated cell aggregation and biofilm formation, which could impact environmental, industrial and medical microbiology.